Recent genetic analyses have shown an unsuspected role for autophagy in Crohn's disease (CD)1, revealing a coding polymorphism in the autophagy gene ATG16L1 that is associated with increased risk of CD4. However, the biological function of ATG16L1 remains relatively unexplored, and the mechanism whereby the T300A risk polymorphism predisposes to CD is not known. The experiments proposed in this application will yield novel insights into the biology of ATG16L1 T300A and provide clues to novel means to preventing or treating CD. Our overarching hypothesis is that ATG16L1 is required for autophagy and that the ATG16L1 T300A CD risk polymorphism contributes to disease by regulating cell-specific defense responses that are central to maintaining intestinal mucosal homeostasis. To test this hypothesis, we propose to determine the functional effects of ATG16L1 T300A in the cellular response to stress and pro-inflammatory ligands and to examine the effects of autophagy mutations in secretion programs essential for mast cell function and Paneth cell function. Furthermore, we will investigate the molecular determinants that contribute to T300A-associated phenotypes and present a novel approach for analysis of gene regulatory networks that will offer insight into the function of ATG16L1 T300A in CD. To investigate these questions, we will employ several genetic approaches, using two unique mouse models developed in the lab (Atg16l1 T300A knock-in mice and conditional deletion of Atg16l1) as well as primary peripheral blood cells from healthy individuals and CD patients bearing this polymorphism. The preliminary data described demonstrate the feasibility of the specific aims of this proposal. The ultimate aim of these studies is to understand the detailed molecular mechanism of ATG16L1 activation that gives rise to aberrant immunological responses to patients with CD. Answers to this central question will have direct clinical implications. Aim 1) Use perturbational profiling by microbial factors to determine how ATG16L1 T300A is involved in CD susceptibility in the context of interactions with the environment (microbes). Aim 2) Unravel underlying mechanisms of phenotypes associated with ATG16L1 T300A in non-canonical autophagy pathways that involve secretion. Aim 3) Identify the molecular determinants of ATG16L1 T300A that contribute to disease risk in CD patients. Aim 4) Identify ATG16L1 T300A-specific regulatory network modules to gain insights into autophagy- associated phenotypes involved in CD pathogenesis.